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Journal of Surgical Research 157, e47–e54 (2009) doi:10.1016/j.jss.2008.11.006

Low-Density Lipoproteins Oxidized After Intestinal Ischemia/Reperfusion in Rats Ishak Ozel Tekin, M.D.,* Emine Yilmaz Sipahi, M.D.,†,1 Mustafa Comert, M.D., ‡ Serefden Acikgoz, M.D.,§ and Gamze Yurdakan, M.D.{ *Department of Immunology; †Department of Pharmacology; ‡Department of Surgery; §Department of Biochemistry; and {Department of Pathology, Faculty of Medicine, Zonguldak Karaelmas University, Zonguldak, Turkey Submitted for publication August 13, 2008

Background. Intestinal ischemia/reperfusion (I/R) is a complex phenomenon causing destruction of both local and remote tissues, as well as multiple-organ failure. We investigated the role of lipid peroxidation in damage to intestinal, liver, and lung tissues in this pathology. Materials and Methods. The superior mesenteric artery was blocked for 30 min followed by 24 h of reperfusion. Tissues were removed and the presence of oxidized LDL, the activities of the superoxide dismutase enzyme, malondialdehyde levels, and inducible nitric oxide synthase expression were each evaluated in the intestinal, liver, and lung tissues. Results. While there was no staining in the control group tissues, ischemia/reperfusion resulted in positive oxidized LDL staining in all of the I/R test group tissue samples. Inducible nitric oxide synthase expression was significantly increased in the ischemia/reperfusion group tissues. Compared with those of the control group rats, the ischemia/reperfusion group tissues showed significantly higher malondialdehyde levels and lower superoxide dismutase activities. Conclusions. This study demonstrated for the first time that oxidized LDL accumulated in the terminal ileum, liver, and lung tissues after intestinal ischemia/reperfusion. This occurrence (or the presence of oxidized LDL) may be an indicator of ongoing oxidative stress and enhanced lipid peroxidation. Augmentation of inducible nitric oxide synthase expression may play a role in progression of inflammation and LDL oxidation. These data support the hypothesis that cellular oxidative stress is a critical step in

1 To whom correspondence and reprint requests should be addressed at Department of Pharmacology, Zonguldak Karaelmas Universitesi, Tip Fakultesi Egitim Bloklari Farmakoloji A.D. 67600 Kozlu, Zonguldak, Turkey. E-mail: dresipahi@yahoo.com.

reperfusion-mediated injury in both the intestine and end organs, and that antioxidant strategies may provide organ protection in patients with reperfusion injury, at least through affecting interaction with free radicals, nitric oxide, and oxidized LDL. Ó 2009 Elsevier Inc. All rights reserved.

Key Words: lipid peroxidation; oxidized low-density lipoproteins; intestinal ischemia/reperfusion.

INTRODUCTION

Intestinal ischemia/reperfusion (I/R) injury is thought to be a causative mechanism for several gastrointestinal diseases, such as necrotizing enterocolitis and mesenteric insufficiency in the elderly, as well as intestinal dysfunction following bowel transplantation [1–3]. The small intestine specifically is highly sensitive to I/R damage. An intestinal ischemic result decreases oxygen and nutrient delivery and induces tissue damage. Reperfusion of ischemic tissue, although necessary for reparative mechanisms, has been shown to worsen acute ischemic injury via the release of ROS, e.g., superoxide [4, 5]. Oxygen radical formation results in damage to an array of biomolecules found in tissues, including nucleic acids, membrane lipids, enzymes and receptors [6]. Membrane-associated polyunsaturated fatty acids are readily attached by ROS in a process that results in the peroxidation of lipids. Peroxidation of membrane lipids disrupts membrane fluidity and cell compartmentation, which can result in cell lysis. Previous studies have suggested that the intestine is extremely susceptible to I/R injury. The intestine is the richest source of the xanthine dehydrogenase-oxidase

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0022-4804/09 $36.00 Ó 2009 Elsevier Inc. All rights reserved.


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enzyme system, which is needed for the production of ROS. Free radicals not only induce oxidative stress in the body, but also initiate lipid peroxidation and maintain the oxidation of low density lipoproteins (LDL) [7, 8]. One of the major and early lipid peroxidation products is comprised of oxidized low-density lipoproteins (oxLDL). Cell-derived reactive oxygen intermediates can affect lipid metabolism, creating an oxLDL particle that is more atherogenic than the native LDL. OxLDLs lead to foam cell formation, inflammatory reactions and cellular hyperplasia [9, 10]. The effects of mesenteric I/R on lipid peroxidation on the intestinal, liver, and lung tissues in rats have not been thoroughly investigated. In the current study, we investigated the following factors: (1) the presence of oxLDL in intestinal tissues after I/R, (2) the association between oxLDL and inducible nitric oxide synthase (iNOS) expression and superoxide dismutase (SOD) activity in mesenteric I/R pathology in the terminal ileum, and (3) the effects of mesenteric I/R on other organs including the liver and lungs.

Histological Examination For the histopathological examination, the tissues were immersed in 10% formalin and allowed to fix for 2 to 3 d. Cross-sections (10 mm) were processed for standard hematoxylin and eosin staining. These sections were then examined via a light microscope and photographed. The terminal ileum histological examination was processed according to the method described previously [12]. The liver histological examination was processed according to a scoring system, named Knodell’s histologic activity index (HAI), which is based on inflammation, necrosis, fibrosis, and structural changes in the liver [13]. The lung sections were also examined for histopathological changes.

Malondialdehyde Measurement The tissue MDA level was assessed according to the method described by Mihara and Uchiyama [14]. Tissues were homogenized with cold 1.15% KCl to make a 10% homogenate; 1% phosphoric acid (3 mL) and a 0.6% thiobarbituric acid (TBA) aqueous solution (1 mL) were added to 0.5 mL of 10% homogenate. The mixture was heated for 45 min in a boiling water bath. After cooling, 4 mL of n-butanol was added and the resulting solution was mixed vigorously. The butanol phase was separated by centrifugation and absorbance was measured at 535 and 520 nm with a Shimadzu UV 1601 spectrophotometer (Kyoto, Japan). The difference was used as the TBA value. 1,1,3,3-tetraetoxypropane was used as the standard. The concentration of MDA was calculated per tissue weight (nmol MDA/g tissue weight).

Tissue Protein Assay MATERIALS AND METHODS Protocols (Rat Intestinal Ischemia/Reperfusion Model) Experiments were performed on adult Sprague-Dawley rats (of both genders) weighing 200 to 230 g (in the Zonguldak Karaelmas University Research Laboratory). The Ethics Committee of Zonguldak Karaelmas University approved the study protocol. The rats were housed under standard laboratory conditions with 12 hours light/dark cycle and were allowed free access to food and water. During the experimental procedures, the animals were placed in separate cages and the laboratory conditions were maintained at room  temperature (22 C). The procedures and protocols of the study were in accord with our institutional guidelines, which are consistent with those of the Guide for the Care and Use of Laboratory Animals, U.S. National Institute of Health, revised 1985. Fourteen animals were divided into two groups of seven rats each, termed the control group and the superior mesenteric artery (SMA) occlusion group, respectively. The procedure used for inducing SMA occlusion has been described previously [11]. The animals were anesthetized by the administration of intramuscular ketamine (90 mg/kg) and xylazine (9 mg/kg). The abdomen was shaved, and following a midline laparotomy, the SMA was occluded for 30 min with an Aesculap Yasargil clip (catalog no: FD 725; Center Valley, PA). Thereafter, the flow was restored for a period of 24 h. Between surgical interventions, the incision was sutured and covered with plastic wrap to minimize the loss of fluids. In the control group, the mesenteric artery was exposed but not occluded and the animals were followed for 30 min to simulate the ischemic interval. The reperfusion interval was also simulated and the animals were then subjected to the same procedures. The control group animals were also housed individually and given free access to water. In the SMA occlusion group, following reperfusion, terminal ileum, liver and lung tissues were harvested and used for determining LDL oxidation, iNOS enzyme activity, the malondialdehyde (MDA) level, SOD expression, and histology as described. The same analysis procedure was carried out in the sham-operated control group.

Protein concentration in the supernatant fraction was determined utulizing the method of Lowry et al. [15]. Reagent A: 2% Na2CO3 in 0.1N NaOH. Reagent B: 0.5% CuSO4.5H2O in 1% sodium or potassium tartrate. Reagent C: alkaline copper solution; 50 mL of Reagent A was mixed with 1 mL of Reagent B. Reagent E Folin reagent. The Folin Ciocalteau reagent was obtained from Sigma Aldrich Chemie GmbH, Steinheim, Germany); 0.2 mL of the sample and 1 mL of Reagent C were mixed and were left undisturbed for 10 min at room temperature. Then 0.10 mLl reagent E was added and the mixture was left for an additional 30 min. Absorbance was measured at 750 nm with a Shimadzu UV 1601 spectrophotometer. Bovine serum albumin (BSA) from Sigma Co. was used as the protein standard. The protein concentration of the supernatant was calculated.

Superoxide Dismutase Assay The assay for SOD activity involved the inhibition of nitroblue tetrazolium (NBT) reduction with xanthine and xanthine oxidase, which was used as a superoxide generator [16]. The tissues were weighed and 1.15% KCl was added to make a 10% homogenate; 2.45 mL of SOD measurement reactive (40 mL 0.3 mMol/L xanthine, 20 ml 0.6 mMol/L EDTA, 20 mL 150 mMol/L NBT, 12 mL 400 mMol/L Na2CO3, 6 mL 1 g/L BSA) was added to 0.5 mL of the 10% homogenate. At 25 C, 0.05 mL of the xanthine oxidase reagent was added to each tube at 30-second intervals and incubated for 20 minutes. The xanthine oxidase reagent was freshly prepared with ice cold 2 Mol/L NH4SO4 and the final concentration of the xanthine oxidase was 167 U/L. The reaction was then terminated by adding 1 mL of 0.8 mMol/L CuCl2 reagent to each tube at 30-seccond intervals. The absorbance of each sample was measured at 560 nm with a Shimadzu UV 1601 spectrophotometer. The percent inhibition was calculated using the following formula: % inhibition Âź

A blank - A sample 3%100 A blank

One unit of SOD is defined as the amount of protein that inhibits the rate of NBT reduction by 50%. SOD activity was calculated per mg of tissue protein (U/mg protein).


TEKIN ET AL.: LDL OXIDATION IN ISCHEMIA-REPERFUSION Inducible nitric oxide synthase immunohistochemical staining method in tissue biopsy materials were fixed in 10% buffered formaldehyde and 3 to 5 mm sections were prepared from paraffin-embedded tissues. After deparaffinization, tissue sections were boiled in 10 nM citrate buffer pH 6.0 for 10 to 20 min followed by cooling at room temperature for 20 min. Then these sections were incubated with primary antibodies (nitric oxide synthase, inducible-(iNOS) Ab-1 rabbit P-Ab neomarkers, from Biogen Medical, Freemont, CA). All of the samples were immunohistochemically analyzed using the avidin-biotin complex method.

Immunofluorescent Staining Method of the Rat Tissue The presence of oxLDL in the tissue sections of the I/R and the sham-operated rats was evaluated using an immunofluorescent stain ing method. The rat tissues were obtained and stored at –85 C in a deep freeze. The slides were prepared from frozen tissue biopsy sections, which were cut at a 7-micron thickness. The slides were further divided into two groups; one was used for the test and the other for the negative control. Thirty microliters of human polyclonal anti-oxLDL IgG solution (IMMCO Diagnostics, New York, NY) were added only to the test slides as the primary antibody and the control slides were manipulated only with the same amount of phosphate buffered saline solution (PBS). After 30 min of incubation in a humid chamber at room temperature, both the control and the test slides were washed with PBS and 30 mL FITC (fluorescent isothiocyanate)-labeled antihuman Ig G were administered as a conjugate substance. For a further 30 min, the slides were kept and incubated at room temperature and then washed with standard PBS solution. After open-air drying, the slides were examined under fluorescent microscopy at 3200 and 3400 magnifications (LEICA DMRX; Wetzlar, Germany).

Statistical Analysis of Results The results were expressed as means 6 standard deviation (means 6 SD). Comparisons between the groups were made using the Studentâ&#x20AC;&#x2122;s t-test. Values of P < 0.05 were considered statistically significant.

RESULTS The Effects of Mesenteric I/R on Terminal Ileum Tissue

Histological Damage No tissue damage was detected in the control terminal ileum tissue (Fig. 1A). Histopathological changes in the I/R group terminal ileum tissues are shown in Fig. 2A. Tissue Oxidized Low Densitylipoprotein Accumulation and Inducible Nitric Oxide Synthase Expression While we did not observe any positive immunofluorescent staining in the control group terminal ileum (the liver control data is shown as the control tissue immunofluorescent staining sample in Fig. 3A), significant positive immunofluorescent staining was observed in the SMA occlusion group terminal ileum tissue (Fig. 3B). While we did not observe any positive iNOS expression in the control group (data not shown), positive iNOS expression was observed in the terminal ileum

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of the I/R group (Fig. 4A). We observed a strong iNOS staining reaction in the mononuclear inflammatory cell cytoplasm in the lamina propria and a weak reaction in the gland epithelium cytoplasm of the terminal ileum tissue in the I/R group. Tissue MDA Level and SOD Activity The mean MDA and SOD contents of the terminal ileum tissue of both the control and I/R groups are shown in Table 1. The terminal ileum MDA levels were found to be significantly higher and the SOD activities were found to be significantly lower in the SMA occlusion group (P < 0.05). The Effects of Mesenteric I/R on Liver and Lung Tissues

Histological Damage No tissue damage was detected in the control liver and lung tissues (Fig. 1B and C). Histopathological changes in the I/R group are shown in Fig. 2B and C. Tissue Oxidized Low Densitylipoprotein Accumulation and Inducible Nitric Oxide Synthase Expression While we did not observe any positive immunofluorescent staining in the control group (data not shown), significant positive immunofluorescent staining was observed in the SMA occlusion group liver and lung tissues (Fig. 3C and D). While we did not observe any positive iNOS expression in the control group (data not shown), positive iNOS expression was observed in the I/R group tissues. A focal reaction was observed in the hepatocyte cytoplasm in the liver tissue (Fig. 4B). A strong iNOS staining reaction was obtained in the alveolar macrophage cytoplasm in the lung tissue (Fig. 4C). Tissue MDA Levels and SOD Activities The mean MDA and SOD contents of the liver and lung tissues are shown in Table 1. The tissue MDA levels were found to be significantly higher and the tissue SOD activities were found to be significantly lower in the SMA occlusion group in both types of tissue (P < 0.05). DISCUSSION

In the current study, we demonstrated that I/R in the mesenteric artery is associated with histopathological damage in the terminal ileum as well as in the end organs, including the liver and lungs. An increase in iNOS expression and MDA levels, in addition to a decrease in SOD activities and ox-LDL accumulation contributed to this damage.


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FIG. 1. Representative hematoxylin and eosin staining in (A) terminal ileum, (B) liver, and (C) lung tissues of control group (A, H&E 310; B and C, H&E 320).

In the current study, we showed that mesenteric artery occlusion in rats is associated with significant mucosal and submucosal damage in the terminal ileum. The effects of mesenteric I/R on lipid peroxidation in rat tissues have not been thoroughly investigated. This is the first study in which accumulation of oxLDL molecules in the terminal ileum was shown by the fluo-

rescent immunostaining method in rats. Due to the fact that the terminal ileum in the control group of rats did not exhibit any accumulation of oxLDL, we can conclude that oxLDL may have played an important role in the I/R-induced terminal ileum damage. We know that free radicals initiate and maintain oxidation of LDL. When oxidized, the unsaturated fatty acids

FIG. 2. Representative hematoxylin and eosin staining in tissues of SMA occlusion group. (A) terminal ileum; marked subepithelial edema, partial separation of apical cells, and inflammation of lamina propria (B) liver; interlobular focal necrosis in liver tissue (mononuclear inflammatory cell infiltration around the hepatocyte in liver sinusoids) (C) lung; increased fibrous tissue in interstitial area, polymorphonuclear leukocyte infiltration, and alveolar edema (A, H&E 34; B, H&E 310; C, H&E 320).


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FIG. 3. Fluorescent microscopic view of the (A) liver section of control group. (B) Terminal ileum, (C) liver, and (D) lung tissues of SMA occlusion group. Significant positive immunofluorescent staining, which indicates accumulation areas of oxLDL was observed in the SMA occlusion group (indicated with arrows) (A and D3400; B and C 3200).

FIG. 4. Immunohistochemical localization of iNOS expression (nitric oxide synthase, inducible, rabbit Pab, biogen) in (A) terminal ileum, (B) liver, and (C) lung tissues of SMA occlusion group (H&E 340).


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TABLE 1 Mean Malondialdehyde (MDA) Concentration and Superoxide Dismutase (SOD) Activity in Rat Tissues MDA (nmol/g tissue)

Terminal ileum Liver Lung

SOD (U/mg protein)

Control

SMA I/R

Control

SMA I/R

138.9 6 45.6 8.2 6 2.1 47 6 12

282.2 6 36.7* 15.1 6 2.3* 93 6 12*

1.39 6 0.4 0.34 6 0.08 0. 49 6 0.04

0.47 6 0.27a 0.17 6 0.07a 0.30 6 0.03a

*a

P < 0.005 using Studentâ&#x20AC;&#x2122;s t-test. Values are mean 6 S.D.

undergo molecular rearrangement, generating peroxy fatty acids. These readily decompose, particularly in the presence of redox metals such as iron and copper, and generate aldehydes such as MDA and 4-hydroxynonenal, among others [17]. Increased intestinal iNOS expression and decreased SOD activity are consistent with observations made through the course of other studies, that iNOS and superoxide are potent modulators of injury after I/R. INOS, one of three NOS isoforms involved in NO synthesis, is characterized by its cytokine upregulation and the production of high quantities of NO. During inflammatory states, gut-derived NO appears to cause direct mucosal injury with disruption of barrier function and misdistribution of blood flow [18]. INOS inhibitors have been shown to ameliorate intestinal hyperpermeability and improve cell viability in lipopolysaccharide (LPS)-exposed animals [19]. We therefore assumed that iNOS-mediated NO acts as a free-radical cytotoxic molecule and plays an important role in the terminal ileum injury induced by mesenteric I/R. Montalto et al. [20] have shown that specific inhibition of iNOS protects the gut from injury after I/R and that complement may mediate tissue damage during I/R by increasing intestinal iNOS and decreasing the activity and protein levels of SOD. In our study, the increase in the tissue MDA level, an index of lipid peroxidation, and the decrease in the tissue SOD level confirm the previously documented finding that I/R injury to the intestine results in oxidative stress and lipid peroxidation. It can be presumed that concentrations of SOD in the gut progressively decrease with ongoing damage induced by oxidative stress. Deshmukh et al. [21] have shown that overexpression of SOD protects gut tissue from neutrophil infiltration and lipid peroxidation during intestinal I/R. Mesenteric I/R is a well-known event causing both local and remote organ injuries. The liver is well recognized as a target during I/R and inflammatory states [22â&#x20AC;&#x201C;25]. Mediators such as oxygen free radicals, NO, and their reaction product peroxynitrite have been found to be involved in I/R-mediated liver injury. While endogenous NO, produced by activation of constitutive

NOS, protects both hepatocytes and endothelial cells against reperfusion injury, iNOS expression usually occurs after inflammatory responses [24]. Controversy exists regarding the effects of NO on the liver. It appears that NO plays a paradoxical role in liver physiology [23, 26]. Small amounts of NO may have a cytoprotective effect, while conversely, there is an increasing body of evidence indicating that overproduction of NO may damage liver function. Thus, NO may have both cytoprotective and cytotoxic properties depending on the amount and isoform of NOS that causes NO production. Lee and coworkers [26] have shown that iNOS knockout mice develop significantly less hepatic injury than do wild-type mice subjected to I/R. In the current study, we showed that intestinal I/R increased iNOS expression in the liver. We therefore deduced that NO acts as a cytotoxic molecule and plays an important role in the liver tissue damage induced by mesenteric I/R. Excessively produced ROS oxidize membrane lipids, critical cellular proteins, and DNA, resulting in lethal hepatocytic injuries. They can also cause oxidation of fatty residues in the liver. We recently showed for the first time that oxLDL may also play a role in liver tissue damage induced by experimental obstructive jaundice [27] in rats. In the current study, the increased tissue MDA level and the decreased SOD level confirmed the previously documented finding that I/R injury to the intestine results in oxidative stress and lipid peroxidation. The liver is the essential organ involved in the purging of oxidatively modified cytotoxic and atherogenic LDL molecules. This organ provides such effective filtration of these molecules that exogenously administrated oxLDL in rats are completely removed from the circulatory system within a few minutes [28]. Furthermore, endothelial and Kupffer cells in the liver contain specific receptors for oxLDL [27]. At high degrees of oxidation of LDL, it has been shown that affinity for the oxLDL specific binding site is strongly enhanced. This is the first study in which accumulation of oxLDL molecules in the liver was shown by fluorescent immunostaining method in rat mesenteric I/R. Since the rat liver tissues from the control group in this study


TEKIN ET AL.: LDL OXIDATION IN ISCHEMIA-REPERFUSION

did not exhibit any signs of accumulation of oxLDL, we can conclude that oxLDL accumulation may have increased the I/R-induced liver damage. Severe intestinal I/R injury is accompanied not only by an acute local inflammatory response but also by significant pulmonary injury and systemic inflammatory changes. The pulmonary system is the most frequently injured organ in multiorgan disorder syndromes. Subsequent to I/R, pulmonary injury may rapidly progress to respiratory failure and acute respiratory distress syndrome (ARDS) [25, 29]. ROS, such as the superoxide radical, hydrogen peroxide, and the hydroxyl radical, have been implicated in the pathogenesis of I/R injury in the lung [7, 30, 31]. The preceding evidence suggests a causal relationship between ROS liberated from the intestine and a remote lung injury. It has been shown that antioxidant therapy may prevent the development of ARDS. Our study confirms the previously documented finding that mesenteric I/R results in reduced SOD and raised MDA levels in rat lung tissue. Mesenteric I/R resulted in significantly increased iNOS expression in lung tissue suggesting that NO generation by iNOS contributes to I/R-induced lung pathology. Uchida et al. [32] observed that the increased lung vascular permeability elicited by gut I/R was significantly attenuated with the inhibition of inducible NO release. We have recently shown that iNOS-mediated NO is also an important factor for pulmonary edema pathology [33, 34]. We demonstrated for the first time the existence of oxLDL in intact rat lung tissue in an a-naphthylthiourea-induced experimental pulmonary edema model [35]. Oxidation of LDL has been implicated both at the onset of and during atherosclerosis; however, its exact pathophysiological role in the lung remains unclear. This study demonstrated for the first time that oxLDL accumulate in intact rat lung tissue after local extrapulmonary I/R. Oxidized lipoproteins appear to participate in the pathogenesis of several lung disorders such as asthma, acute lung injury, and cystic fibrosis [36]. Zhou et al. [37] have shown that oxLDL inhibit surfactant phosphatidylcholine synthesis in murine lung epithelial cells and may injure the lung by modifying the surfactant. OxLDL have been suggested as modulators of the inflammation associated with asthma. In vivo activation of cutaneous mast cells and release of histamine enhance the transendothelial transport of plasma LDL [38]. In turn, oxLDL can cause mast cell degranulation and increased leukocyte rolling, adhesion, and migration. Under certain conditions, oxLDL can promote vascular permeability and dilation, thus leading to further edema and leukocyte adhesion. OxLDL can also stimulate vasoconstriction and inhibit smooth muscle and endothelial-dependent relaxation [39].

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In conclusion, our study is the first description of oxLDL accumulation in the terminal ileum as well as in the end organs, including the liver and lungs after 30 min ischemia/24 h reperfusion of the SMA in rats. An increase in iNOS expression and MDA levels and a decrease in SOD enzyme activities participated in this injury. These results support the conclusion that I/R injury may extend beyond the ischemic area at risk to include injury to remote, nonischemic organs and that oxidative and nitrosative stress may play a major role in tissue damage. ACKNOWLEDGMENTS The authors thank Hasan Tahsin Yilmaz for his assistance in the research laboratory.

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Low-Density Lipoproteins Oxidized After Intestinal IschemiaReperfusion